Regulatory T cells contribute to the prolonged survival of allogeneic tissues under some conditions. Many investigators have characterized regulatory cells by their phenotypic markers (such as CD4+CD25+) and others have tried to determine the mechanisms by which they work. In studies of allogeneic cardiac transplantation in mice using costimulatory blockade, we found that prolonged survival could not be achieved in recipients that lacked MHC class II molecules. This suggested that regulatory T cells might be generated through the indirect pathway. We have now accumulated additional preliminary data, using an adoptive transfer system, indicating that donor-specific CD4+CD25+ regulatory cells are specific for indirect determinants. In addition, the data are consistent with a model in which: 1) Peptides of donor MHC antigens are primarily responsible for generating the indirect determinants for this regulatory population, which functions by preventing activation of donor-reactive lymphocytes in secondary lymphoid organs. 2) An additional population of self-specific, CD4+ regulatory T cells is also be generated that can prevent the effector function of donor-reactive lymphocytes, but not their activation, if the self-determinant is expressed in the donor graft. 3) CD8+ cells alter the regulatory response, perhaps by producing additional populations of regulatory T cells. The experiments proposed in this application will focus on the specificity of regulatory T cells in transplantation, which will enable us to separate distinct subpopulations of these cells. We will test our proposed model by pursuing three specific aims that examine the different regulatory T cell subpopulations. The experiments in the fourth specific aim will use the information about their specificity to compare mechanistic properties of the regulatory T cell subpopulations and to develop diagnostic assays to detect their presence.